Previous work has demonstrated that unique isoforms of nonmuscle myosin heavy chain-B (MHC-B) are expressed in chicken and human neuronal cells (Takahashi et al., J. Biol. Chem. 267: 17864, 1992). These isoforms, which are generated by alternative splicing of pre-mRNA, differ from the MHC-B isoform present in a large number of nonmuscle cells in that they contain inserted cassettes of amino acids near the ATP binding region and/or near the actin binding region. The insert near the ATP binding region begins after amino acid 211 and consists of either 10 or 16 amino acids and contains a putative phosphorylatable serine for proline- directed kinases. The insert near the actin binding region begins after amino acid 621 and consists of 21 amino acids. We have studied the distribution and expression of the inserted MHC-B isoforms. In the developing chicken brain, mRNA encoding the 10 amino acid insert gradually increases after fertilization, peaks in the 10-14 day embryo and then declines. In contrast, the mRNA encoding the 21 amino acid insert appears just before birth and is abundantly expressed in the adult cerebellum. There is a marked species difference between the distribution of the inserted isoforms in adult tissues. The 10 amino acid insert near the ATP binding region is very poorly expressed in chicken brain, but makes up most of the MHC-B mRNA expressed in human cerebrum and approximately 90% of MHC-B mRNA in human retina. The 21 amino acid insert is abundantly expressed in chicken cerebellum and human cerebrum, but is absent from human retina and human cultured cells. Using a variety of neuronally derived cell lines, we studied conditions that increase the expression of the 10 amino acid insert present near the ATP binding region. Employing human retinoblastoma (Y-79) and neuroblastoma (SK-N-SH) cell lines, we are able to increase the expression of the inserted isoform using a number of agonists or serum deprivation. In each case, expression of the inserted isoform correlates with cell differentiation and inhibition of cell division. Using nerve growth factor and a rat pheochromocytoma cell line (PC12), we are able to show that the appearance of the inserted isoform correlates temporally and reversibly with neurite outgrowth. Using bovine brain myosin and immunoprecipitated nonmuscle MHC-B from a butyrate-treated retinoblastoma (Y-79) cell extract, we are able to phosphorylate the unique site, present in the inserted cassette of amino acids, with proline-directed kinases.